Article Figures & Data

Figures

Knockdown of Ubc9 impairs integrin-mediated T cell adhesion and LFA-1 clustering. (A and B) Stable Ubc9-knockdown Jurkat T cells (shUbc9#1 and shUbc9#2) or control shEGFP Jurkat T cells (shEGFP) were either left untreated or stimulated with anti-CD3, followed by the measurement of cell adhesion to ICAM-1–coated plates (A) and fibronectin-coated plates (B) as described in Materials and Methods. Error bars indicate the SE from three individual experiments for the ICAM-1–binding assay and four individual experiments for the fibronectin-binding assay. Immunoblots on the right panels show anti-Ubc9 Western blot of whole cell lysates of cells used in the ICAM-1–/fibronectin-binding assays. (C and D) Murine primary T cells infected with lentiviral-encoded shEGFP or shUbc9#2 were stimulated with anti-CD3 and assessed for adhesion to ICAM-1–coated plates (C) and fibronectin-coated plates (D). Error bars indicate the SE from three individual experiments. (E) Upper panel: Stable Ubc9-knockdown and control shEGFP Jurkat T cells were stimulated with anti-CD3 for 30 min and imaged for LFA-1 clustering as described in Materials and Methods. The arrow designates LFA-1 cluster; scale bars, 10 μm. Lower panel: Histogram showing the percentage of T cells with LFA-1 clustering. Error bars indicate the SE from four individual experiments. (F) Representative FACS profile showing the integrin LFA-1 α and β subunits and TCR surface expression levels in the stable Ubc9-knockdown Jurkat T cells (shUbc9#2) and in mock control T cells (shEGFP). Data are representative of at least three independent experiments. *p < 0.05, **p < 0.01.

Ubc9 binds directly to the immune adaptor ADAP but not SKAP1 in vivo and in vitro. (A) Left panel: Mouse hybridoma DC27.10 T cells were preactivated with anti-CD3 (2C11) for 5 min and prepared for immunoprecipitation with either IgG control, anti-ADAP, anti-SKAP1, or anti-RanGAP1 followed by blotting with anti-ADAP (top) and anti-Ubc9 Ab (bottom). Right panel: Relative bound Ubc9 to each Ab was calculated as the band intensity by ImageJ and was plotted as fold difference relative to the anti-CD3–stimulated IgG control. (B) Upper panel: The interaction of Ubc9 with ADAP in primary murine T cells. Primary murine CD4+ T cells were stimulated with anti-CD3 for the indicated time periods. Endogenous ADAP was immunoprecipitated with anti-ADAP Ab or IgG control, followed by immunoblotting with Abs against ADAP or Ubc9. Lower panel: Time course of Ubc9 binding to ADAP following anti-CD3 stimulation. 2B4 cells were stimulated with anti-CD3 for the indicated time periods. Endogenous ADAP was immunoprecipitated with anti-ADAP Ab and assayed for interaction with Ubc9 by immunoblotting with Abs against ADAP or Ubc9. (C) HEK 293T cells were transfected with Ubc9 alone, or together with either HA-ADAP, HA-SKAP1, HA-ADAP dNLS2, or HA-RanGAP1 constructs. Immunoprecipitation was performed using anti-HA Ab, followed by blotting with anti-HA (top) or anti-Ubc9 Ab (bottom). The black lines indicate where parts of the image were joined. (D) Detection of Ubc9 in anti-ADAP immunoprecipitates using mass spectrometry analysis. Left panel: Coomassie brilliant blue staining analysis of anti-CD3–stimulated Jurkat T cell lysate prepared for immunoprecipitation with IgG control and anti-ADAP. Bands indicated with rectangle were excised and subjected to liquid chromatography tandem mass spectrometry analysis as described in Materials and Methods. Right panel: List of detected peptides assigned to Ubc9 and their abundance. (E) The putative NLS domains in ADAP. Schematic drawing of domain structure of ADAP and the ADAP NLS1 deletion mutant (ADAP dNLS1), and NLS2 deletion mutant (ADAP dNLS2) constructs (upper panel). The corresponding sequences of both NLSs are listed and compared (lower panel). NLS1 and NLS2 share an identical lysine-containing sequence of KKFK (in bold), and both have several positively charged residues as KR/KK that resemble the bipartite nuclear localization motif (in shaded). A unique 5-aa motif of KKLKK (in bold and underlined) is present in NLS2 but not NLS1, which is also found in the Ubc9-interacting NLSs of AR and Vsx-1 proteins (26, 50). (F) HEK 293T cells were cotransfected with Ubc9 and vector control, ADAP WT, ADAP dNLS1, or ADAP dNLS2, followed by precipitation with anti-HA Ab and immunoblotting with anti-Ubc9 (top) and anti-ADAP Ab (bottom).

ADAP-Ubc9 binding fails to affect TCR proximal signaling events and TCR-induced IL-2 transcription. (A) Stable Ubc9-knockdown and control shEGFP Jurkat T cells were either left untreated or stimulated with anti-CD3 (OKT3) for 3 min. The cell lysates were extracted and subjected to Western blotting with Abs against phosphotyrosine, α-tubulin, and Ubc9. (B) Jurkat T cells were cotransfected with IL-2 (upper left panel) or NF-κB (upper right panel) promoter-driven luciferase reporter with either shEGFP or shUbc9#2 constructs and were stimulated with anti-CD3 for 6 h, followed by a measurement of luciferase activity as indicated in Materials and Methods. Error bars indicate the SE from three individual experiments. The knockdown of Ubc9 was assessed by anti-Ubc9 Western blotting in the lower panel. (C) Stable Ubc9-knockdown Jurkat T cells (shUbc9#2) or the mock control T cells (shEGFP) were either left unstimulated or were stimulated with anti-CD3 for 5 min. Whole cell lysate was subjected to immunoprecipitation with anti–ZAP-70 (left panel) and anti–PLC-γ1 (right panel), followed by Western blotting with indicated Abs. (D) Stable Ubc9-knockdown Jurkat T cells (shUbc9#2) or the mock control cells (shEGFP) were loaded with indo-1. Intracellular Ca2+ flux after anti-CD3 or ionomycin stimulation was measured by flow cytometry. Arrows indicate the time of addition of stimulus. (E) Stable Ubc9-knockdown Jurkat T cells (shUbc9#2) or the mock control cells (shEGFP) were cultured with anti-CD3 for 24 h, stained with FITC-CD69, and analyzed by FACS. (F) Left panel: Jurkat T cells were cotransfected with SKAP1 and either ADAP WT, ADAP dNLS1, or ADAP dNLS2 constructs, followed by immunoprecipitation with anti-SKAP1 Ab and blotting with anti-ADAP and anti-SKAP1. Right panel: COS-7 cells were cotransfected with SLP-76 and either ADAP WT, ADAP dNLS1, or ADAP dNLS2 constructs, followed by immunoprecipitation with anti–SLP-76 Ab and immunoblotting with anti-ADAP and anti–SLP-76. (G) Jurkat T cells were cotransfected with IL-2 promoter-driven luciferase reporter and either vector, ADAP WT, ADAP dNLS1, or ADAP dNLS2 constructs, stimulated with anti-CD3 for 6 h followed by luciferase activity analysis as described in Materials and Methods. Error bars indicate the SE from three individual experiments. (H) HA-tagged ADAP WT and deletion mutants were expressed in Jurkat T cells and stimulated with ionomycin plus PMA for 30 min. Confocal images of the cells showed subcellular localizations of ADAP WT and deletion mutants (green) by immunofluorescence microscopy using anti-HA Ab. Scale bar, 5 μm. ns, not significant.

Effects of Ubc9 knockdown on LFA-1–mediated membrane localization of Rap1-RIAM-RapL and fibronectin-mediated Rac1 activation in response to anti-CD3 stimulation. (A) Purified mouse primary CD4+ cells were infected with lentiviruses expressing shRNAs against Ubc9 or EGFP, and cultured ex vivo for 2 d. Cells were either left unstimulated or stimulated with 4 μg/ml anti-CD3 (2C11) for 10 min, and subjected to cytosolic and plasma membrane fractionation, followed by Western blotting with Abs against Rap1, RapL, RIAM, tubulin, and Ubc9 as indicated. The anti-tubulin blotting was used as an internal control. (B) Double immunofluorescence staining of Rap1 (green) and LFA-1 (red) in stable Ubc9-knockdown or control shEGFP Jurkat T cells with or without anti-CD3 stimulation. Cells were fixed, permeabilized, stained, and visualized for LFA-1 (red) and Rap1 (green) as described in the Materials and Methods. Images (Ba–Bd) are representative of three independent experiments. Scale bar, 5 μm. The graph (Be) indicates the percentage of Rap1 localized at the plasma membrane from a total of 30–50 cells in each condition. (C) Stable Ubc9-knockdown Jurkat T cells (shUbc9#2) or the mock control T cells (shEGFP) were either left unstimulated or stimulated with 5 μg/ml anti-CD3 (OKT3) for 60 min. Rac1 activity was assessed by pull-down assays using GST-PBD-PAK. GTP-bound Rac1 and total Rac1 were determined by immunoblot with anti-Rac1 Ab. Right panel: the bar graph represented the relative intensity of the bands (Rac-GTP/Total Rac1) measured by ImageJ software. The fold induction of each sample was normalized to that of unstimulated shEGFP cells. Error bars indicate the SE from three individual experiments. ****p < 0.0001.

Proposed model for the selective role of Ubc9-ADAP in the regulation of TCR-mediated T cell adhesion and signaling. ADAP possesses regulatory roles in the integrin inside-out signaling for T cell adhesion and in TCR signaling events. The ADAP-SKAP1 module regulates the activation of integrin LFA-1 via its downstream two parallel signaling axis: TCR-Rap1-RapL-LFA-1 αL and Rap1-RIAM-Talin-LFA-1 β2 (10, 43, 60). The distinct pool of ADAP-SKAP independently associates with RapL and RIAM, but collectively determines the integrin LFA-1 activation (22). In addition, the ADAP-SKAP1 module facilitates VLA-4 activation via signaling axis TCR-Rac1-VLA-4/fibronectin (45–47). Our data suggest a model for the selective role of Ubc9-ADAP in the TCR-induced integrin activation for T cell adhesion. In this model, TCR stimulation induces Ubc9 association with ADAP. Although it has no effect on the TCR proximal signaling responses and TCR-induced IL-2 transcription, the interaction of Ubc9 with ADAP is selectively required for TCR-induced Rac1 activation that leads to the enhancements of VLA-4–mediated T cell adhesion to fibronectin (signaling axis 2 in circle), and the membrane targeting of Rap1 and RapL, but not RIAM for the activation of LFA-1 integrin (signaling axis 1 in circle). Thus, our findings reconfigure the existing model of inside-out signaling and introduce the Ubc9-ADAP module as a new regulatory layer on the controls of integrin-mediated T cell adhesion.